Cell surface ATP receptors can be divided into the metabotropic receptor family (P2Y/P2U) and the ionotropic receptor family (or the P2xc3x97 receptor family). Metabotropic receptor family members are G-protein coupled receptors and ionotropic receptor family members are ligand-gated channels. There are eleven metabotropic receptor family members and seven ionotropic receptor family members, P2xc3x971R to P2xc3x977R.
The P2xc3x977 receptor (P2xc3x977R), like other members of the P2xc3x97 receptor family, is an ATP-gated ion channel (Surprenant et al. (1996) Science 272:735-738; Rassendren et al. (1997) J. Biol. Chem. 272:5482-5486; Michel et al. (1998) Br. J. Pharmacol. 125:1194-1201). The P2xc3x977R, however, demonstrates attributes that clearly distinguish it from other members of the family. For example, the P2xc3x977R requires levels of ATP in excess of 1 mM to achieve activation whereas other P2xc3x97 receptors activate at ATP concentrations xe2x89xa6100 xcexcM (Greenberg et al. (1988) J. Biol. Chem. 263:10337-10343; Steinberg et al. (1987) J. Biol. Chem. 262:8884-8888): the higher concentration requirement reflects, in part, the preference of the P2xc3x977R for ATP4xe2x88x92 as its ligand and the relatively low abundance of this species in media containing physiological concentrations of divalent cations (e.g., Ca2+ and Mg2+). An additional unique feature of the P2xc3x977R is found in its conductance properties. All P2xc3x97 receptors demonstrate non-selective channel-like properties following ligation, but the channels formed by the P2xc3x977R rapidly transform to xe2x80x9cporesxe2x80x9d that allow passage of solutes as large as 900 daltons (Steinberg et al. (1987) J. Biol. Chem. 262:8884-8888; Virgihio et al. (1999) J. Physiol. 519:335-346). Molecular details of this transformation remain to be described, but domain swapping and deletion experiments have suggested that the carboxy terminal domain of the P2xc3x977R participates in pore complex formation; the carboxy terminal domain is significantly longer than the comparable domains in the other P2xc3x97 receptors (North (1996) Current Opin. Cell Biol. 8:474-483). Possibly as a consequence of this pore-like activity, continuous ligation of the P2xc3x977 receptor for times greater than 15 minutes can lead to cell death (Di Virgilio (1995) Immunol. Today 16:524-528; Murgia et al. (1992) Biochem. J. 288:897-901; Ferrari et al. (1999) FEBS Let. 447:71-75).
The P2xc3x977R displays a restricted cellular distribution, being observed primarily in cells of hematopoietic origin including monocytes and macrophages and some lymphocyte populations (Di Virgilio (1995) Immunol. Today 16:524-528; Collo et al. (1997) Neuropharmacol. 36:1277-1283). The receptor also has been reported to exist on microglial cells (Sanz et al. (2000) J. Immunol. 164:4893-4898), some cancer cells (Wiley et al. (1989) Blood 73:1316-1323), sperm (Foresta et al. (1996) Am J. Physiol. 270:C1709-C1714), and dendritic cells (Mutini et al. (1999) J. Immunol. 163:1958-1965).
The P2xc3x977R has been reported to participate in a diverse list of cellular activities including lymphocyte proliferation (Baricordi et al. (1999) J. Biol. Chem. 274:33206-33208), fertilization (Foresta et al. (1996) Am J. Physiol. 270:C1709-C1714), giant cell formation (Chiozzi et al. (1997) J. Cell Biol. 138:697-706), cell death (Murgia et al. (1992) Biochem. J. 288:897-901; Ferrari et al. (1999) FEBS Let. 447:71-75), killing of invading mycobacteria (Latumas et al. (1997) Immunity 7:433-444), and IL-1 posttranslational processing (Hogquist et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:8485-8489; Perregaux et al. (1994) J. Biol. Chem. 269:15195-15203). Further, ligation of the P2xc3x977R has been associated with activation of phospholipase D and activation of some forms of NF-xcex5B (Humphreys et al. (1996) J. Immunol. 157:5627-5637; Ferrari et al. (1997) J. Cell Biol. 139:1635-1643).
One of the most intriguing activities attributed to the P2xc3x977R is its ability to induce posttranslational processing of proIL-1 (Sanz et al. (2000) J. Immunol. 164:4893-4898; Hogquist et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:8485-8489; Perregaux et al. (1994) J. Biol. Chem. 269:15195-15203). Interleukin (IL)-1 is a multipotential inflammatory mediator produced in abundance by activated monocytes and macrophages. The administration of IL-1 to animals has been shown to initiate an inflammatory response, produce fever, and promote tissue degradation. Further, elevated levels of IL-1 have been detected in patients suffering from a number of chronic disorders, including rheumatoid arthritis, Alzheimer""s disease, and acute myelocytic leukemia (McNiff et al. (1995) Cytokine 7:209; Gray et al. (1986) J. Immunol. 137:3644; Lomedico et al. (1984) Nature 312:458).
When IL-1 is released from cells, it binds to receptors on target cells and elicits complex signaling cascades leading to the upregulation of gene products that contribute to an inflammatory state including matrix metalloproteinases, cyclooxygenase-2, IL-6, and cellular adhesion molecules (Flannery et al. (1999) Matrix Biol. 18:225-237; Guzn et al. (1998) J. Biol. Chem. 273:28670-28676; Allen et al. (2000) J. Exp. Med. 191:859-869; Bevilacqua et al. (1989) Science 243:1160-1164). Two distinct gene products, IL-1xcex1 and IL-1xcex2, contribute to IL-1 biological activity. The amino acid sequences of IL-1a and IL-1xcex2 are only 25% identical yet these two polypeptides bind to the same receptors on target cells (Slack et al. (1993) J. Biol. Chem. 268:2513-2524). Human EL-1xcex1 and IL-xcex2 are both initially produced as 31 kDa procytokines containing amino terminal extensions that are subsequently removed by proteolysis. In the case of proIL-1xcex1, the propolypeptide and the 17 kDa cleavage product display equivalent signaling activity indicating that proteolytic cleavage is not necessary to generate a receptor-competent ligand. In contrast, proIL-1xcex2 does not bind to the signaling IL-1 receptor (Mosley et al. (1987) J. Biol. Chem. 262:2941-2944), and cleavage by caspase-1 is necessary to generate the mature 17 kDa signaling-competent form of this cytokine (Cerretti et al. (1992) Science 256:97-100; Thornberry et al. (1992) Nature 356:768-774).
The two forms of IL-1 share another very unusual attribute, both proIL-1xcex1 and proIL-1xcex2 are synthesized without a signal sequence (March et al. (1985) Nature 315:641-647), the peptide epitope required to direct nascent polypeptides to the endoplasmic reticulum (Walter et al. (1994) Ann. Rev. Cell Biol. 10:87-119). As a result, newly synthesized proIL-1xcex1 and proIL-1xcex2 accumulate within the cytoplasmic compartment of producing cells rather than being sequestered to the secretory apparatus. Caspase-1 also is produced as a cytosol-localized proenzyme, the 45 kDa propolypeptide must be proteolytically processed to generate the 20 kDa and 10 kDa subunits which constitute the mature active protease (Thornberry et al. (1992) Nature 356:768-774; Miller et al. (1993) J. Biol. Chem. 268:18062-18069; Ayala et al. (1994) J. Immunol. 153:2592-2599). In activated monocytes and macrophages, therefore, proIL-1xcex2 and procaspase-1 co-exist within the cytoplasm. Mechanisms that control activation of procaspase-1, and in turn cleavage of proIL-1xcex2, are not well understood. Recent studies, however, have provided evidence that proteolytic processing IL-1xcex2 and release of the mature cytokine product extracellularly do not proceed constitutively. Rather, the post-translational processing of proIL-1 requires that lipopolysaccharide-(LPS)-activated monocytes and/or macrophges encounter an external stimulus that promotes activation of procaspase-1, cleavage of proIL-1xcex2, and release of the 17 kDa cytokine (Miller et al. (1995) J. Immunol. 154:1331-1338; Laliberte et al. (1999) J. Biol. Chem. 274:36944-36951; Sanz et al. (2000) J. Immunol. 164:4893-4898). Stimuli that function in vitro to promote IL-1 posttranslational processing by LPS-activated monocytes and/or macrophages include ATP, nigericin, cytolytic T-cells, bacterial toxins and hypotonic stress (Bhakdi et al. (1990) J. Clin. Invest. 85:7988-7992; Hogquist et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:8485-8489; Perregaux et al. (1992) J. Immunol. 149:1294-1303; Perregaux et al. (1994) J. Biol. Chem. 269:15195-15203; Perregaux et al. (1996) J. Immunol. 157:57-64; Walev et al. (1995) EMBO J. 14:1607-1614). This requirement for secretion stimulus is not restricted to cells in culture; mouse peritoneal macrophages produce proIL-1xcex2 in response to intraperitoneal (i.p.) injection of LPS, but release little cytokine extracellularly (Griffiths et al. (1995) J. Immunol. 154:2821-2828). Subsequent i.p. injection of ATP, however, stimulates generation of large quantities of extracellular mature IL-1xcex2 (Griffiths et al. (1995) J. Immunol. 154:2821-2828).
The present invention provides non-human, genetically- modified mammals and animal cells containing a disrupted P2xc3x977R gene that prevents or reduces endogenous P2xc3x977R function.
In one aspect, the invention provides a genetically-modified, non-human mammal, wherein the genome of the mammal comprises a genetic modification to a copy of the P2xc3x977R gene that results in disrupted P2xc3x977R gene function from the modified gene. In one embodiment, the mammal is heterozygous for the modified P2xc3x977R gene. In another embodiment, the mammal is homozygous for the modified P2xc3x977R gene.
In preferred embodiments, the mammal is a rodent, such as a mouse. In some embodiments, the mouse is homozygous for the modified P2xc3x977R gene and exhibits reduced ATP-stimulated intracellular translocation of macromolecules. In other embodiments, the mouse is homozygous for the modified P2xc3x977R gene and exhibits reduced ATP-stimulated interleukin-(IL)-1xcex1, IL-1xcex2, or IL-18 posttranslational processing or reduced ATP-stimulated IL-6 production.
In some embodiments, the P2xc3x977R gene is disrupted by homologous recombination, and in other embodiments, the P2xc3x977R gene is disrupted by the insertion of a gene trapping vector.
The invention provides, in another aspect, a method for producing a genetically-modified mouse comprising a disrupted P2xc3x977R gene. In this method, a nucleic acid molecule is introduced into a mouse embryonic stem cell, wherein the nucleic acid molecule inserts into the P2xc3x977R gene, thereby disrupting the gene. In one embodiment of the method of the invention, the nucleic acid molecule disrupts the P2xc3x977R gene by homologous recombination. In another embodiment, the nucleic acid molecule disrupts the P2xc3x977R gene by the insertion of a gene trapping vector. The mouse embryonic stem cell containing the introduced nucleic acid is then introduced into a mouse embryo, which is then transplanted into a pseudopregnant mouse. The embryo containing the introduced mouse embryonic stem cell is allowed to develop to term, after which a genetically-modified mouse is identified whose genome comprises a disruption of a P2xc3x977R gene.
In another aspect, the invention provides an isolated nucleic acid molecule comprising a P2xc3x977R gene targeting construct. Upon introduction of the targeting construct into a cell, the construct recombines with a P2xc3x977R gene in the cellular genome, thereby inserting itself at the P2xc3x977R gene locus and disrupting P2xc3x977R gene function in the cell.
The invention provides, in yet another aspect, a genetically-modified animal cell comprising a genetic modification in at least one copy of P2xc3x977R gene in the genome of the cell, the genetic modification resulting in disrupted function in the modified copy of P2xc3x977R gene. In some embodiments, the cell is a mammalian cell. In a preferred embodiment, the cell is an embryonic stem cell. In some embodiments, the cell is derived from a genetically-modified, non-human mammal or non-human mammalian embryo. In some embodiments, the cell is heterozygous for the modified P2xc3x977R gene, and in other embodiments the cell is homozygous for the modified P2xc3x977R gene.
Various terms used herein are defined as stated below.
By a non-human mammal or an animal cell that is xe2x80x9cgenetically-modifiedxe2x80x9d is meant such a mammal or cell having a modification in a gene of interest which was introduced into the non-human mammal or cell, or a progenitor mammal or cell, by genetic engineering. The non-human mammal or cell is heterozygous or homozygous for the modified gene. In a genetically-modified, non-human mammal, it is preferred that all somatic cells and germline cells contain the modified gene.
This modification disrupts P2xc3x977R gene function and features the insertion of a foreign nucleic acid sequence into the P2xc3x977R gene locus, either alone or in combination with a deletion of the endogenous P2xc3x977R gene sequence, and the modification can occur within any region of the P2xc3x977R gene, e.g., in an enhancer, promoter, regulator region, noncoding region, coding region, intron, or exon.
By a nucleic acid sequence that is xe2x80x9cexogenousxe2x80x9d or xe2x80x9cforeignxe2x80x9d to a P2xc3x977R gene is meant a sequence that is non-naturally occurring in the P2xc3x977R gene. Portions of the exogenous sequence may be either xe2x80x9chomologousxe2x80x9d or xe2x80x9cheterologousxe2x80x9d to P2xc3x977R gene. By xe2x80x9chomologousxe2x80x9d is meant a sequence that is related to a P2xc3x977R gene to a degree sufficient to allow in vivo hybridization. By contrast, a xe2x80x9cheterologousxe2x80x9d sequence is unrelated to P2xc3x977R gene and does not hybridize in vivo with a P2xc3x977R gene.
By xe2x80x9cdisrupted P2xc3x977R gene functionxe2x80x9d is meant a decrease in the P2xc3x977R polypeptide activity encoded by the modified P2xc3x977R gene. When the genetic modification in a non-human mammal or animal cell effectively eliminates all wild type copies of the P2xc3x977R gene (e.g., the non-human mammal or animal cell is homozygous for the P2xc3x977R gene disruption or the only wild type copy of the P2xc3x977R gene originally present in the genome of the non-human mammal or animal cell is now disrupted), then the P2xc3x977R gene disruption results in a statistically significant reduction in the P2xc3x977R polypeptide activity, as compared to the wild type, non-human mammal or animal cell of the same strain or type. This reduction in P2xc3x977R polypeptide activity results from reduced expression of the P2xc3x977R gene (i.e., reduced P2xc3x977R mRNA levels produce reduced levels of P2xc3x977R polypeptide) and/or because the modified P2xc3x977R gene encodes a mutated polypeptide with reduced function compared to a wild type P2xc3x977R polypeptide. Preferably, the activity of the P2xc3x977R polypeptide is reduced to about 50% or less of wild type levels, more preferably, to about 25% or less, and, even more preferably, to about 10% or less of wild type levels. Most preferably, the P2xc3x977R polypeptide activity is nondetectable in the genetically modified, non-human mammal or animal cell (i.e., the P2xc3x977R gene disruption results in a null mutation).
By xe2x80x9cpseudopregnantxe2x80x9d mouse is meant a female mouse with hormone levels comparable to a pregnant mouse and sufficient to maintain a pregnancy. A pseudopregnant mouse is prepared by mating a female mouse in natural estrus with a vasectomized or genetically sterile male mouse.
By xe2x80x9creducedxe2x80x9d is meant a statistically significant decrease (i.e., p less than 0.1).
By xe2x80x9cmodulatesxe2x80x9d is meant a statistically significant increase or decrease in level.
By xe2x80x9cP2xc3x977R polypeptide activityxe2x80x9d or xe2x80x9cP2xc3x977R-like activityxe2x80x9d is meant an increased ATP-stimulated cell permeability and an increased accumulation of a macromolecule, e.g., a dye macromolecule such as YoPro Yellow, an increase in ATP-stimulated IL-1xcex1, IL-1xcex2, or IL-18 posttranslational processing in activated inflammatory cells (e.g., activated by lipopolysaccharide treatment or cytokine treatment, such as tumor necrosis factor-xcex1), an increase in ATP-stimulated IL-6 production in inflammation-induced cells, or lymphoproliferation.
The term xe2x80x9canimal cellxe2x80x9d is meant to include a cell in an immortalized cell line, in a primary cell preparation, or otherwise derived from an animal. Preferably, the animal is a mammal such as a human, mouse, or rat.